细粒棘球蚴EG95s重组蛋白串联表达免疫原性分析

Immunogenicity of tandem expression recombinant EG95s protein of Echinococcus granulosus

目的比较、分析EG95s重组蛋白的免疫原性。方法本研究分别诱导表达含有1、2、3拷贝EG95s的pET-1EG95s、pET-2EG95s和pET-3EG95s重组质粒,并用His亲和纯化HIS-1EG95s、HIS-2EG95s和HIS-3EG95s3种重组蛋白。再以相同的免疫程序分别免疫8周龄BALB/c小鼠,通过间接ELISA方法检测小鼠体内抗体水平的变化分析其免疫原性。结果经过改造的EG95s重组蛋白:HIS-1EG95s、HIS-2EG95s、HIS-3EG95s均保留了免疫原性,能诱导小鼠产生特异性抗体。3者抗体水平的比较结果显示HIS-1EG95s首次免疫后1周产生的抗体水平明显高于HIS-2EG95s、HIS-3EG95s,但免疫后2周开始,HIS-3EG95s抗体水平超过HIS-1EG95s组,并在二次免疫及三次免疫后持续高于HIS-1EG95s组。免疫后最终抗体效价显示HIS-1EG95s和HIS-2EG95s两组均为1∶819 200,而HIS-3EG95s组为1∶1 638 400。HIS-1EG95s、HIS-2EG95s、HIS-3EG95s均诱导机体产生IFN-γ,但差异不显著,与羊包虫阳性血清反应结果显示HIS-1EG95s的反应性均显著低于HIS-2EG95s和HIS-3EG95s。结论 HIS-1EG95s、HIS-2EG95s与HIS-3EG95s均具有很好的免疫原性,虽HIS-EG95s在免疫初期免疫效果好,但串联3拷贝的EG95s后使得重组蛋白免疫效果更持久,更有利于产生更长效的免疫保护作用。本研究为羊包虫病疫苗的研制及免疫预防奠定了科学基础。

In order to analyze the immunogenicity of the recombinant EG95s protein ,the recombinant plasmids of pET-1EG95s ,pET-2EG95s and pET-3EG95s which containing respectively 1 ,2 ,and 3 copies EG95s were induced to express HIS-1EG95s ,HIS-2EG95s and HIS-3EG95s ,and then the proteins were purified and identified by western-blotting .The same im-mune process was used ,and 8 weeks-old BALB/c mice were immunized ,then its immunogenicity was analyzed by detecting an-tibody levels in mice by indirect ELISA method .Results showed that for recombinant EG95s proteins after transformation , HIS-1EG95s ,HIS-2EG95s ,and HIS-3EG95s also retained immunogenicity and could induce specific antibodies in mice .One week＇s late after the first immunization with HIS-1EG95s ,the antibody level of was significantly higher than HIS-2EG95s and HIS-3EG95s .But began from 2 weeks after immunization ,the antibody level of HIS-3EG95s was always higher than that of HIS-1EG95s group during the period of the immune .Both the final antibody titers after immunization of HIS-1EG95s and HIS-2EG95s groups was 1∶819 200 ,while HIS-3EG95s group was 1∶163 840 0 .HIS-1EG95s ,HIS-2EG95s and HIS-3EG95s all induced IFN-γin immune mice ,but the difference was not significant .The HIS-1EG95s showed lower response to Echinococ-cus granulosus positive serum than HIS-2EG95s and HIS-3EG95s .It’s indicated that the HIS-1EG95s and HIS-3EG95s also had good immunogenicity .HIS-3EG95s make recombinant protein immunic effects more lasting ,and benefit to generate more long-lasting protective immunity .This study provides the scientific basis for the immunization of echinococcosis （hydatidosis） .